The role of bidirectional communication between the adipokines and the endogenous opioid system in an experimental mouse model of colitis-associated colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of death globally. Multiple factors may contribute to the pathogenesis of CRC, including the abnormalities in the functioning of the endogenous opioid system (EOS) or adiponectin-related signaling. The aim of our study was to evaluate if differences in the expression of opioid receptors (ORs) influence the development of CRC and if modulation of adiponectin receptors using AdipoRon, a selective AdipoR1 receptor agonist, affects colorectal carcinogenesis. METHODS: Naltrexone, an opioid receptor antagonist, was injected intraperitoneally every second day for 2 weeks, at the dose of 1 mg/kg in healthy Balb/C mice to induce changes in ORs expression. CRC was induced by a single intraperitoneal injection of azoxymethane (AOM) and the addition of dextran sodium sulfate (DSS) into drinking water in three-week cycles. The development of CRC was assessed using macro- and microscopic scoring and molecular analysis (RT qPCR, ELISA) after 14 weeks. RESULTS: Naltrexone significantly increased the mRNA expression of Oprm1, Oprd1, and Oprk1 in the mouse colon and in the brain (non-significantly). The pretreatment of mice with naltrexone aggravated the course of CRC (as indicated by tumor area, colon thickness, and spleen weight). The level of circulatory adiponectin was lowered in mice with CRC and increased in the colon as compared with healthy mice. The ß-endorphin level was increased in the plasma of mice with CRC and decreased in the colon as compared to healthy mice. AdipoRon, AdipoR1 agonist, worsened the CRC development, and pretreatment with naltrexone enhanced this negative effect in mice. CRC did not affect the expression of the Adipor1 gene, but the Adipor1 level was increased in mice pretreated with naltrexone (AOM/DSS and healthy mice). AdipoRon did not influence the expression of opioid receptors at the mRNA level in the colon of mice with CRC. The mRNA expression of Ptgs2, Il6, Nos2, Il1b, Il18, Gsdmd, and Rela was increased in mice with CRC as compared to the healthy colon. AdipoRon significantly decreased mRNA expression of Ptgs2, Il6, Il1b, and Il18 as compared to CRC mice. CONCLUSION: EOS and adiponectin-related signaling may play a role in the pathogenesis of CRC and these systems may present some additivity during carcinogenesis.

Empagliflozin attenuates intestinal inflammation through suppression of nitric oxide synthesis and myeloperoxidase activity in in vitro and in vivo models of colitis.

Inflammatory bowel diseases (IBD) are characterized by chronic and relapsing inflammation affecting the gastrointestinal (GI) tract. The incidence and prevalence of IBD are relatively high and still increasing. Additionally, current therapeutic strategies for IBD are not optimal. These facts urge todays' medicine to find a novel way to treat IBD. Here, we focused on the group of anti-diabetic drugs called gliflozins, which inhibit sodium glucose co-transporter type 2 (SGLT-2). Numerous studies demonstrated that gliflozins exhibit pleiotropic effect, including anti-inflammatory properties. In this study, we tested the effect of three gliflozins; empagliflozin (EMPA), dapagliflozin (DAPA), and canagliflozin (CANA) in in vitro and in vivo models of intestinal inflammation. Our in vitro experiments revealed that EMPA and DAPA suppress the production of nitric oxide in LPS-treated murine RAW264.7 macrophages. In in vivo part of our study, we showed that EMPA alleviates acute DSS-induced colitis in mice. Treatment with EMPA reduced macro- and microscopic colonic damage, as well as partially prevented from decrease in tight junction gene expression. Moreover, EMPA attenuated biochemical inflammatory parameters including reduced activity of myeloperoxidase. We showed that SGLT-2 inhibitors act as anti-inflammatory agents independently from their hypoglycemic effects. Our observations suggest that gliflozins alleviate inflammation through their potent effects on innate immune cells.

ABCG2 Gene and ABCG2 Protein Expression in Colorectal Cancer-In Silico and Wet Analysis.

ABCG2 (ATP-binding cassette superfamily G member 2) is a cell membrane pump encoded by the ABCG2 gene. ABCG2 can protect cells against compounds initiating and/or intensifying neoplasia and is considered a marker of stem cells responsible for cancer growth, drug resistance and recurrence. Expression of the ABCG2 gene or its protein has been shown to be a negative prognostic factor in various malignancies. However, its prognostic significance in colorectal cancer remains unclear. Using publicly available data, ABCG2 was shown to be underexpressed in colon and rectum adenocarcinomas, with lower expression compared to both the adjacent nonmalignant lung tissues and non-tumour lung tissues of healthy individuals. This downregulation could result from the methylation level of some sites of the ABCG2 gene. This was connected with microsatellite instability, weight and age among patients with colon adenocarcinoma, and with tumour localization, population type and age of patients for rectum adenocarcinoma. No association was found between ABCG2 expression level and survival of colorectal cancer patients. In wet analysis of colorectal cancer samples, neither ABCG2 gene expression, analysed by RT-PCR, nor ABCG2 protein level, assessed by immunohistochemistry, was associated with any clinicopathological factors or overall survival. An ABCG2-centered protein-protein interaction network build by STRING showed proteins were found to be involved in leukotriene, organic anion and xenobiotic transport, endodermal cell fate specification, and histone methylation and ubiquitination. Hence, ABCG2 underexpression could be an indicator of the activity of certain signalling pathways or protein interactors essential for colorectal carcinogenesis.

Assessment of the TGFB1 gene expression and methylation status of the promoter region in patients with colorectal cancer.

The aim of this study was to evaluate the expression of the TGFB1 gene encoding the TGF-ß1 cytokine in 64 patients, and then to compare it with clinico-pathological features. The study also investigated whether the regulation of the gene expression is caused by methylation of the promoter region between - 235 and + 22 nucleotide from the start of transcription. The dependence of the relative level of the TGFB1 gene expression on the clinical advancement according to the TNM classifications was shown. Additionally, the individual grades of the T and M features of the TNM classification differed in the relative transcript levels of the TGFB1 gene. Moreover, the higher relative expression level of the studied gene was associated with a lack of vascular invasion by cancer cells and presence of lymphocytes in the neoplastic tissue. The obtained results may indicate a possible impact of the gene on the process of carcinogenesis in colorectal cancer and reduction of its expression level may be one of the factors contributing to progression of the disease.

FGF7/FGFR2-JunB signalling counteracts the effect of progesterone in luminal breast cancer.

We have recently demonstrated that fibroblast growth factor receptor 2 (FGFR2)-mediated signalling alters progesterone receptor (PR) activity and response of oestrogen receptor α (ER)-positive (ER+) breast cancer (BCa) cell lines to anti-ER agents. Little is known about whether the crosstalk between ER and PR, shown to be modulated by the hormonal background, might also be affected by FGFR2. Here, PR-dependent behaviour of ER+ BCa cells was studied in the presence of oestrogen (E2) and progesterone (P4) and/or FGF7. In vitro analyses showed that FGF7/FGFR2 signalling: (a) abolished the effect of P4 on E2-promoted 3D cell growth and response to tamoxifen; (b) regulated ER and PR expression and activity; (c) increased formation of ER-PR complexes; and (d) reversed P4-triggered deregulation of ER-dependent genes. Analysis of clinical data demonstrated that the prognostic value of FGFR2 varied between patients with different menopausal status; that is, high expression of FGFR2 was significantly associated with longer progression-free survival (PFS) in postmenopausal patients, whereas there was no significant association in premenopausal patients. FGFR2 was found to positively correlate with the expression of JunB proto-oncogene, AP-1 transcription factor subunit (JUNB), an ER-dependent gene, only in premenopausal patients. Molecular analyses revealed that the presence of JunB was a prerequisite for FGFR2-mediated abrogation of P4-induced inhibition of cell growth. Our results demonstrate for the first time that the FGF7/FGFR2-JunB axis abolishes the modulatory effects of PR on ER-associated biological functions in premenopausal ER+ BCa. This may provide foundations for a better selection of patients for FGFR-targeting therapeutic strategies.

The Anti-Inflammatory Effect of Acidic Mammalian Chitinase Inhibitor OAT-177 in DSS-Induced Mouse Model of Colitis.

Inflammatory bowel diseases (IBD) are chronic and relapsing gastrointestinal disorders, where a significant proportion of patients are unresponsive or lose response to traditional and currently used therapies. In the current study, we propose a new concept for anti-inflammatory treatment based on a selective acidic mammalian chitinase (AMCase) inhibitor. The functions of chitinases remain unclear, but they have been shown to be implicated in the pathology of various inflammatory disorders regarding the lung (asthma, idiopathic pulmonary fibrosis) and gastrointestinal tract (IBD and colon cancer). The aim of the study is to investigate the impact of AMCase inhibitor (OAT-177) on the dextran sulfate sodium (DSS)-induced models of colitis. In the short-term therapeutic protocol, OAT-177 given intragastrically in a 30 mg/kg dose, twice daily, produced a significant (p < 0.001) anti-inflammatory effect, as shown by the macroscopic score. Additionally, OAT-177 significantly decreased TNF-α mRNA levels and MPO activity compared to DSS-only treated mice. Intraperitoneal administration of OAT-177 at a dose of 50 mg/kg caused statistically relevant reduction of the colon length. In the long-term therapeutic protocol, OAT-177 given intragastrically in a dose of 30 mg/kg, twice daily, significantly improved colon length and body weight compared to DSS-induced colitis. This is the first study proving that AMCase inhibitors may have therapeutic potential in the treatment of IBD.

Characterization of the Synergistic Effect between Ligands of Opioid and Free Fatty Acid Receptors in the Mouse Model of Colitis.

BACKGROUND: Recent studies suggest that lipids, including free fatty acids (FFAs), are necessary for proper µ opioid receptor (MOR) binding and that activation of opioid receptors (ORs) improves intestinal inflammation. The objective of the study was to investigate a possible interaction between the ORs and FFA receptors (FFARs) ligands in the colitis. METHODS: The potential synergistic effect of ORs and FFARs ligands was evaluated using mouse model of acute colitis induced by dextran sulfate sodium (DSS, 4%). Compounds were injected intraperitoneally (i.p.) once or twice daily at the doses of 0.01 or 0.02 mg/kg body weight (BW) (DAMGO-an MOR agonist), 0.3 mg/kg BW (DPDPE-a δ OR (DOR) agonist) and 1 mg/kg BW (naloxone-a non-selective OR antagonist, GLPG 0974-a FFAR2 antagonist, GSK 137647-a FFAR4 agonist and AH 7614-a FFAR4 antagonist) for 4 days. RESULTS: Myeloperoxidase (MPO) activity was significantly decreased after DAMGO (0.02 mg/kg BW) and GSK 137647 (1 mg/kg BW) administration and co-administration as compared to DSS group. CONCLUSIONS: Treatment with ligands of ORs and FFARs may affect the immune cells in the inflammation; however, no significant influence on the severity of colitis and no synergistic effect were observed.

Intertumoral Heterogeneity of Primary Breast Tumors and Synchronous Axillary Lymph Node Metastases Reflected in IHC-Assessed Expression of Routine and Nonstandard Biomarkers.

Breast cancer (BC) remains a significant healthcare challenge. Routinely, the treatment strategy is determined by immunohistochemistry (IHC)-based assessment of the key proteins such as estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67. However, it is estimated that over 75% of deaths result from metastatic tumors, indicating a need to develop more accurate protocols for intertumoral heterogeneity assessment and their consequences on prognosis. Therefore, the aim of this preliminary study was the identification of the expression profiles of routinely used biomarkers (ER, PR, HER2, Ki-67) and additional relevant proteins [Bcl-2, cyclin D1, E-cadherin, Snail+Slug, gross cystic disease fluid protein 15 (GCDFP-15), programmed death receptor 1 (PD-L1), and phosphatase of regenerating liver 3 (PRL-3)] in breast primary tumors (PTs) and paired synchronous axillary lymph node (ALN) metastases. A total of 67 tissue samples met the inclusion criteria for the study. The expression status of biomarkers was assessed in PTs and ALN metastases using tissue microarrays followed by IHC. In 11 cases, the shift of intrinsic molecular BC subtype was noticed between PTs and paired ALN metastases. Moreover, a significant disproportion in E-cadherin presence (p = 0.0002) was noted in both foci, and the expression status of all proteins except for HER2 demonstrated considerable variance (k = 1, p < 0.0001). Importantly, in around 30% of cases, the ALN metastases demonstrated discordance, i.e., loss/gain of expression, compared to the PTs. Intertumoral synchronous heterogeneity in both foci (primary tumor and node metastasis) is an essential phenomenon affecting the clinical subtype and characteristics of BC. Furthermore, a greater understanding of this event could potentially improve therapeutic efficacy.

MicroRNA profile and iron-related gene expression in hepatitis C-related hepatocellular carcinoma: a preliminary study.

INTRODUCTION: Hepatocellular carcinoma (HCC) is very difficult to diagnose, especially in its early stages. Non-invasive diagnostic and prognostic factors for this cancer are urgently needed. The purpose of our study was to investigate whether the microRNAs (miRNAs) regulating genes involved in iron homeostasis, whose disruption is a hallmark of HCC, offer potential as diagnostic or prognostic factors of HCV-related hepatocellular carcinoma. MATERIAL AND METHODS: Serum and tumor samples, and adjacent liver specimens, were obtained from 65 HCC patients. Additionally, serum samples were obtained from 65 healthy controls. In total, 28 circulating and eight tissue microRNA expression profiles were estimated by TaqMan qPCR. RESULTS: The expression profiles of all tested miRNAs were altered in the hepatocellular carcinoma patients. Iron level was negatively related to serum miR-96 level in healthy controls. Although the expression of iron metabolism proteins correlated with the level of serum miRNA in the controls, this was not observed in cancer patients. In the group of cancer patients, Let-7a, miR-29b, and miR-133a were positively related to ferroportin, transferrin and ferritin levels, while miR-31, miR-221 and miR-532 were negatively related to ferroportin, transferrin receptor 1 and ferritin levels. According to ROC curve analyses, 15 miRNAs are able to discriminate with 100% sensitivity and specificity between hepatocellular carcinoma patients and healthy subjects, which is more efficient than α-fetoprotein. CONCLUSIONS: Circulating miRNAs that regulate the expression of iron metabolism proteins should be evaluated as promising candidates for HCV-related HCC diagnostic agents.

Activation of Free Fatty Acid Receptor 4 Affects Intestinal Inflammation and Improves Colon Permeability in Mice.

Diet is considered an important trigger in inflammatory bowel diseases (IBD), as feeding habits can affect intestinal permeability and clearance of bacterial antigens, consequently influencing the immune system. Free fatty acid receptors (FFARs), expressed on the intestinal epithelial cells, belong to the family of luminal-facing receptors that are responsive to nutrients. The objective of this study was to characterize the anti-inflammatory activity and the effect on intestinal barrier function of synthetic FFAR agonists in mouse models of colitis. Therapeutic activity of GW9508 (FFAR1 agonist), 4-CMTB (FFAR2 agonist), AR420626 (FFAR3 agonist), and GSK137647 (FFAR4 agonist) was investigated in two models of semi-chronic colitis: induced by trinitrobenzenesulfonic acid (TNBS), mimicking Crohn's disease, as well as induced by dextran sulfate sodium (DSS), which recapitulates ulcerative colitis in humans. Moreover, we assessed the influence of FFARs agonists on epithelial ion transport and measured the ion flow stimulated by forskolin and veratridine. Administration of FFAR4 agonist GSK137647 attenuated both TNBS-induced and DSS-induced colitis in mice, as indicated by macroscopic parameters and myeloperoxidase activity. The action of FFAR4 agonist GSK137647 was significantly blocked by pretreatment with selective FFAR4 antagonist AH7614. Moreover, FFAR1 and FFAR4 agonists reversed the increase in the colon permeability caused by inflammation. FFAR4 restored the tight junction genes expression in mouse colon. This is the first evaluation of the anti-inflammatory activity of selective FFAR agonists, showing that pharmacological intervention targeting FFAR4, which is a sensor of medium and long chain fatty acids, attenuates intestinal inflammation.

AdipoRon, an Orally Active, Synthetic Agonist of AdipoR1 and AdipoR2 Receptors Has Gastroprotective Effect in Experimentally Induced Gastric Ulcers in Mice.

INTRODUCTION: Adiponectin is a hormone secreted by adipocytes, which exhibits insulin-sensitizing and anti-inflammatory properties and acts through adiponectin receptors: AdipoR1 and AdipoR2. The aim of the study was to evaluate whether activation of adiponectin receptors AdipoR1 and AdipoR2 with an orally active agonist AdipoRon has gastroprotective effect and to investigate the possible underlying mechanism. METHODS: We used two well-established mouse models of gastric ulcer (GU) induced by oral administration of EtOH (80% solution in water) or diclofenac (30 mg/kg, p.o.). Gastroprotective effect of AdipoRon (dose 5 and 50 mg /kg p.o) was compared to omeprazole (20 mg/kg p.o.) or 5% DMSO solution (control). Clinical parameters of gastroprotection were assessed using macroscopic (gastric lesion area) and microscopic (evaluation of the gastric mucosa damage) scoring. To establish the molecular mechanism, we measured: myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities; glutathione (GSH) level; and IL-1ß, adenosine monophosphate-activated protein kinase (AMPK), and phosphorylated AMPK expression in gastric tissue. RESULTS: AdipoRon produced a gastroprotective effect in both GU mouse models as evidenced by significantly lower macroscopic and microscopic damage scores. AdipoRon exhibited anti-inflammatory effect by reduction in MPO activity and IL-1ß expression in the gastric tissue. Moreover, AdipoRon induced antioxidative action, as demonstrated with higher GSH levels, and increased SOD and GPX activity. CONCLUSIONS: Activation of AdipoR1 and AdipoR2 using AdipoRon reduced gastric lesions and enhanced cell response to oxidative stress. Our data suggest that AdipoR1 and AdipoR2 activation may be an attractive therapeutic strategy to inhibit development of gastric ulcers.

New Class of Anti-Inflammatory Therapeutics Based on Gold (III) Complexes in Intestinal Inflammation-Proof of Concept Based on In Vitro and In Vivo Studies.

Inflammatory bowel diseases (IBD) are at the top of the worldwide rankings for gastrointestinal diseases as regards occurrence, yet efficient and side-effect-free treatments are currently unavailable. In the current study, we proposed a new concept for anti-inflammatory treatment based on gold (III) complexes. A new gold (III) complex TGS 121 was designed and screened in the in vitro studies using a mouse macrophage cell line, RAW264.7, and in vivo, in the dextran sulphate sodium (DSS)-induced mouse model of colitis. Physicochemical studies showed that TGS 121 was highly water-soluble; it was stable in water, blood, and lymph, and impervious to sunlight. In lipopolysaccharide (LPS)-stimulated RAW264.7 cells, the complex showed a potent anti-inflammatory profile, as evidenced in neutral red uptake and Griess tests. In the DSS-induced mouse model of colitis, the complex administered in two doses (1.68 µg/kg, intragastrically, and 16.8 µg/kg, intragastrically, once daily) produced a significant (* p < 0.05) anti-inflammatory effect, as shown by macroscopic score. The mechanism of action of TGS 121 was related to the enzymatic and non-enzymatic antioxidant system; moreover, TGS 121 induced changes in the tight junction complexes expression in the intestinal wall. This is the first study proving that gold (III) complexes may have therapeutic potential in the treatment of IBD.

Assessment of the Role of Selected SMAD3 and SMAD4 Genes Polymorphisms in the Development of Colorectal Cancer: Preliminary Research.

BACKGROUND: Colon cancer is one of the most common types of malignant tumor worldwide. The molecular mechanism of colorectal carcinogenesis is very complex and not yet fully understood. The TGFß (transforming growth factor ß) signaling pathway plays a significant role in the development of many cancers, including colorectal cancer pathogenesis. Changes in TGFß pathway are associated with increased colorectal cancer risk, because this pathway participates in the control of important cellular processes such as cell growth, proliferation, differentiation, or apoptosis. The family of SMAD (similar to mother against decapentaplegic) proteins is closely correlated to this pathway. SMADs genes expression affects modulation of the transcription of many genes, which leads to the inhibition of cell-growth and apoptosis in colon epithelial cells. The presence of SNPs (single nucleotide polymorphisms) in SMADs genes encoding proteins involved in the control of biological processes important for the cell may play a significant role in the predisposition to the development of colorectal cancer, or in the regulation of the severity of changes related to tumor growth. Extension of data in this field may provide clinically significant conclusions influencing the implementation of personalized treatment based on specific changes characteristic of a patient with colorectal cancer. PURPOSE: The subject of this research was genotyping polymorphisms of SMAD3 (rs6494629) and SMAD4 (rs10502913, rs12968012, rs1057520801) genes in the group of patients with colorectal cancer and in the control group, and comparing the genotypic frequency distributions with clinical-pathological features within the study group and between the groups. MATERIALS AND METHODS: SNP genotyping analysis was performed on genomic DNA isolated from 84 frozen tissue sections of colorectal cancer and from 60 peripheral blood samples of patients without cancer. To evaluate the polymorphic variants of SMAD genes, the restricted fragment length of a polymorphism reaction (PCR-RFLP) was used. RESULTS: The results obtained in the study showed no significant association between the examined polymorphisms and the risk of developing colorectal cancer. CONCLUSION: More extensive studies to confirm the results obtained in this study are needed. Further studies on a larger study group divided according to the clinical stage and histological differentiation may allow finding or excluding the significance of the studied SNPs as potential markers of colorectal cancer in relation to the clinico-pathological data.

Serous borderline tumor with distant mediastinal metastasis? An Exceptional presentation of ovarian tumor.

Borderline ovarian tumor is a non-invasive lesion with an excellent prognosis. Here we report a case of 48-year-old woman with distinctive clinical presentation of metastasis of ovarian adenocarcinoma, which was an microinvasive component of a serous borderline tumor. On initial diagnosis patient did not present any clinical manifestation of ovarian tumor. Histological examination of resected ovary showed typical features of the serous borderline tumor with one very diminutive focus of invasive serous adenocarcinoma 4mm in diameter. This exceptional case shows that borderline tumors of ovary with any features of invasion could present an aggressive course with distant metastases.

Novel selective agonist of GPR18, PSB-KK-1415 exerts potent anti-inflammatory and anti-nociceptive activities in animal models of intestinal inflammation and inflammatory pain.

BACKGROUND: GPR18 is a recently deorphanized receptor which was reported to act with several endogenous cannabinoid ligands. Here, we aimed to describe the role of GPR18 in intestinal inflammation and inflammatory pain. METHODS: The anti-inflammatory activity of selective GPR18 agonist, PSB-KK-1415, and antagonist, PSB-CB5, was characterized in semi-chronic and chronic mouse models of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). The extent of inflammation was evaluated based on the macroscopic and microscopic scores, quantification of myeloperoxidase (MPO) activity, and Western blot analyses of tumor necrosis factor-α (TNF-α) and interleukin-6 in colonic tissue. The expression of GPR18 in colonic samples from patients with Crohn's disease (CD) was quantified using real-time PCR. The anti-nociceptive potential of the agonist in intestinal inflammation was evaluated in the mouse model of inflammatory pain. KEY RESULTS: In semi-chronic colitis, PSB-KK-1415 reduced macroscopic score (1.79 ± 0.22 vs. 2.61 ± 0.48), expression of TNF-α (1.89 ± 0.36 vs. 2.83 ± 0.64), and microscopic score (5.00 ± 0.33 vs. 6.45 ± 0.40), all compared to mice with colitis. In chronic colitis, PSB-KK-1415 decreased macroscopic score (3.33 ± 1.26 vs. 4.00 ± 1.32) and MPO activity (32.23 ± 8.51 vs. 41.33 ± 11.64) compared to inflamed mice. In the mouse model of inflammatory pain, PSB-KK-1415 decreased the number of pain-induced behaviors in both, controls (32.60 ± 2.54 vs. 58.00 ± 6.24) and inflamed mice (60.83 ± 2.85 vs. 85.00 ± 5.77) compared to animals without treatment with PSB-KK-1415 (P < 0.005 for both). Lastly, we showed an increased expression of GPR18 in CD patients compared to healthy controls (3.77 ± 1.46 vs. 2.38 ± 0.66, p = 0.87). CONCLUSIONS & INFERENCES: We showed that GPR18 is worth considering as a potential treatment target in intestinal inflammation and inflammatory pain.

Anti-inflammatory and antibacterial effects of human cathelicidin active fragment KR-12 in the mouse models of colitis: a novel potential therapy of inflammatory bowel diseases.

INTRODUCTION: Inflammatory bowel diseases (IBD) are a group of chronic gastrointestinal tract disorders with complex etiology, with intestinal dysbiosis as the most prominent factor. In this study, we assessed the anti-inflammatory and antibacterial actions of the human cathelicidin LL-37 and its shortest active fragment, KR-12 in the mouse models of colitis. MATERIALS AND METHODS: Mouse models of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and dextran sulfate sodium (DSS) were used in the study. The extent of inflammation was evaluated based on the macro- and microscopic scores, quantification of myeloperoxidase (MPO) activity and microbiological analysis of stool samples. RESULTS: A preliminary study with LL-37 and KR-12 (1 mg/kg, ip, twice daily) showed a decrease in macroscopic and ulcer scores in the acute TNBS-induced model of colitis. We observed that KR-12 (5 mg/kg, ip, twice daily) reduced microscopic and ulcer scores in the semi-chronic and chronic TNBS-induced models of colitis compared with inflamed mice. Furthermore, qualitative and quantitative changes in colonic microbiota were observed: KR-12 (5 mg/kg, ip, twice daily) decreased the overall number of bacteria, Escherichia coli and coli group bacteria. In the semi-chronic DSS-induced model, KR-12 attenuated intestinal inflammation as demonstrated by a reduction in macroscopic score and colon damage score and MPO activity. CONCLUSIONS: We demonstrated that KR-12 alleviates inflammation in four different mouse models of colitis what suggests KR-12 and cathelicidins as a whole are worth being considered as a potential therapeutic option in the treatment of IBD.

A 'Real-Life' Experience on Automated Digital Image Analysis of FGFR2 Immunohistochemistry in Breast Cancer.

We present here an assessment of a 'real-life' value of automated machine learning algorithm (AI) for examination of immunohistochemistry for fibroblast growth factor receptor-2 (FGFR2) in breast cancer (BC). Expression of FGFR2 in BC (n = 315) measured using a certified 3DHistech CaseViewer/QuantCenter software 2.3.0. was compared to the manual pathologic assessment in digital slides (PA). Results revealed: (i) substantial interrater agreement between AI and PA for dichotomized evaluation (Cohen's kappa = 0.61); (ii) strong correlation between AI and PA H-scores (Spearman r = 0.85, p < 0.001); (iii) a small constant error and a significant proportional error (Passing-Bablok regression y = 0.51 × X + 29.9, p < 0.001); (iv) discrepancies in H-score in cases of extreme (strongest/weakest) or heterogeneous FGFR2 expression and poor tissue quality. The time of AI was significantly longer (568 h) than that of the pathologist (32 h). This study shows that the described commercial machine learning algorithm can reliably execute a routine pathologic assessment, however, in some instances, human expertise is essential.

Upregulation of HIF1-α via an NF-κB/COX2 pathway confers proliferative dominance of HER2-negative ductal carcinoma in situ cells in response to inflammatory stimuli.

There are data to suggest that some ductal carcinoma in situ (DCIS) may evolve through an evolutionary bottleneck, where minor clones susceptible to the imposed selective pressure drive disease progression. Here, we tested the hypothesis that an impact of the inflammatory environment on DCIS evolution is HER2-dependent, conferring proliferative dominance of HER2-negative cells. In tissue samples, density of tumour-infiltrating immune cells (TIICs) was associated only with high tumour nuclear grade, but in 9% of predominantly HER2-negative cases, the presence of tumoral foci ('hot-spots') of basal-like cells with HIF1-α activity adjacent to the areas of dense stromal infiltration was noted. Results of in vitro analyses further demonstrated that IL-1ß and TNF-α as well as macrophage-conditioned medium triggered phosphorylation of NF-κB and subsequent upregulation of COX2 and HIF1-α, exclusively in HER2-negative cells. Treatment with both IL-1ß and TNF-α resulted in growth stimulation and inhibition of HER2-negative and HER2-positive cells, respectively. Moreover, ectopic overexpression of HIF1-α rescued HER2-positive cells from the negative effect of IL-1ß and TNF-α on cell growth. Our data provide novel insight into the molecular basis of HER2-dependent proliferation of DCIS cells and indicate the NF-κB/COX2 → HIF1-α signalling axis as a dominant mechanism of DCIS evolution induced by inflammatory microenvironment. Presented findings also highlight the clinical significance of heterogeneity of DCIS tumours and suggest that HIF1-α might be considered as a predictive marker of disease progression.

Hormonal Receptor Status Determines Prognostic Significance of FGFR2 in Invasive Breast Carcinoma.

Interaction between fibroblast growth factor receptor 2 (FGFR2) and estrogen/progesterone receptors (ER/PR) affects resistance to anti-ER therapies, however the prognostic value of FGFR2 in breast cancer (BCa) remains largely unexplored. We have recently showed in vitro that FGFR2-mediated signaling alters PR activity and response to anti-ER treatment. Herein, prognostic significance of FGFR2 in BCa was evaluated in relation to both ER/PR protein status and a molecular signature designed to reflect PR transcriptional activity. FGFR2 was examined in 353 BCa cases using immunohistochemistry and Nanostring-based RNA quantification. FGFR2 expression was higher in ER+PR+ and ER+PR- compared to ER-PR- cases (p < 0.001). Low FGFR2 was associated with higher grade (p < 0.001), higher Ki67 proliferation index (p < 0.001), and worse overall and disease-free survival (HR = 2.34 (95% CI: 1.26-4.34), p = 0.007 and HR = 2.22 (95% CI: 1.25-3.93), p = 0.006, respectively). The poor prognostic value of low FGFR2 was apparent in ER+PR+, but not in ER+PR- patients, and it did not depend on the expression level of PR-dependent genes. Despite the functional link between FGFR2 and ER/PR revealed by preclinical studies, the data showed a link between FGFR2 expression and poor prognosis in BCa patients.

Cyclic derivative of morphiceptin Dmt-cyclo-(D-Lys-Phe-D-Pro-Asp)-NH2(P-317), a mixed agonist of MOP and KOP opioid receptors, exerts anti-inflammatory and anti-tumor activity in colitis and colitis-associated colorectal cancer in mice.

Endogenous opioid system is involved in the maintenance of the intestinal homeostasis. Recently, we proved that stimulation of opioid receptors using P-317, a cyclic morphiceptin analog, resulted in the alleviation of acute colitis in mice. The aim of the current study was to assess the effect of P-317 during colitis and colitis-associated colorectal cancer in mice. Colitis was induced by addition of dextran sodium sulfate (DSS) into drinking water. Colitis-associated colorectal cancer was induced by a single intraperitoneal injection of azoxymethane (AOM) and subsequent addition of DSS into drinking water (week 2, 5, 8). During macroscopic damage evaluation the samples were collected and used for biochemical (MPO activity assay), molecular (qPCR and western blot) and histological studies. In experimental colitis, P-317 induced an anti-inflammatory response as indicated by macroscopic and microscopic scores. In the colitis-associated colorectal cancer model, a significant difference in colorectal tumor development was observed between vehicle- and P-317-treated mice. P-317 decreased the total number of colonic tumors and inhibited MPO activity. Hematoxylin and eosin staining confirmed anti-tumor activity of P-317. The expression of TNF-α was decreased in P-317-treated mice as compared to the vehicle-treated group. P-317 decreased proliferation as well as ß-catenin expression in tumors. P-317, a mixed MOP and KOP receptor agonist, induced an anti-inflammatory response in experimental colitis and decreased tumor development in colitis-associated colorectal cancer in mice.